小麦内源基因 GAG56D的快速检测技术体系

为了满足转基因小麦检测中对内源参照基因的检测要求,以小麦属特异的内源基因γ-醇溶蛋白基因（ GAG56D）作为目的基因,采用环状等温扩增技术(LAMP)建立了小麦内源基因 GAG56D的快速检测技术体系。针对小麦内源基因 GAG56D特异靶序列的6个区域设计4 条特异性引物,通过显色法进行LAMP检测,并对时间、温度等反应条件及反应灵敏度、特异性、稳定性进行探索。结果表明,该检测体系最适反应温度为63℃,反应时间 60 min,定性检测低限达到0.5%（w/w）。该检测方法具有高度的特异性、灵敏度和稳定性,操作简单、快速。     

Preliminary investigation and detection based on loop-mediated isothermal amplification (LAMP) of phytoplasmas associated with diseases in B. napus L.

In the last decade, some disease occurred on our experimental farms that had caused serious losses. They were not caused by fungi, bacteria or viruses. By loop-mediated isothermal amplification (LAMP) technique, the detection results pointed to the possible pathogen as phytoplasma. The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B. napus L., including witches’ broom, multi-stems, aggregate main inflorescences, and flat stems. The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61% in 2010 to 6.00% in 2021. Some phytoplasma infected plants died without any growing points. These studies would be helpful for detecting phytoplasmas diseases, selecting disease resistant germplasm and improving varieties with disease resistances in B. napus L.     

Screening of three sweet potato (Ipomoea batatas L.) cultivars for resistance to different virulence groups of root-knot nematodes (Meloidogyne spp.) under controlled conditions

The susceptibility of three sweet potato cultivars (Ipomoea batatas L.) C4, TIS 3290 and TIS 9162 was evaluated against 156 isolates of Meloidogyne spp. with the aim to include resistant/tolerant sweet potato cultivars in a crop rotation scheme for the management of root-knot nematodes. The nematode isolates corresponded to races 1, 2 and 3 of Meloidogyne arenaria (n = 7), races 1, 2, 3 and 4 of M. incognita (n = 131) and Meloidogyne javanica (n = 18). Also, the isolates of M. incognita were differentiated in virulence groups: Pepper (n = 35), Pepper-Mi (n = 25), Tomato (n = 41) and Tomato-Mi (n = 30), depending on their ability to parasitize resistant pepper and tomato cultivars. The tested isolates of M. javanica parasitized C4 and TIS 3290, but not TIS 9162, whereas M. arenaria parasitized C4 and TIS 9162, but not TIS 3290, and M. incognita was able to parasitize the three sweet potato cultivars tested. C4 was the most susceptible cultivar to all nematode species tested, especially M. incognita, TIS 3290 was the most resistant and TIS 9162 was in between (7.2, 62.9 and 26.9% of resistant plants, respectively). Susceptibility of the sweet potato cultivars showed slight variations depending on the race or virulence group of M. incognita. The results suggest that sweet potato cultivars TIS 3290 and TIS 9162 may be used as rotation crops in fields where root-knot nematodes are present, their selection depending on the Meloidogyne isolates present. The use of resistant sweet potato cultivars would be preferably combined with other management practices to avoid virulence selection in nematode isolates.     

Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms

The nematicidal effect of Pseudomonas fluorescens, Paecilomyces lilacinus, Pichia guilliermondii and Calothrix parietina singly or in combination was tested against root-knot nematode, Meloidogyne incognita. Treatments with P. fluorescens and P. lilacinus caused mortality of M. incognita as 45% and 30% of juveniles after 48 h of exposures, respectively compared to water control in vitro. Under greenhouse conditions, all treatments reduced the disease severity and enhanced plant growth compared to untreated control. Application of P. fluorescens, P. lilacinus and P. guilliermondii Moh 10 was more effective compared to C. parietina. There was a negative interaction between C. parietina and either P. lilacinus or P. guilliermondii. Fresh and dry weight of shoots and roots of plants were significantly reduced as a result of infection with M. incognita, however application of biocontrol agents singly or in mix recovered this reduction. Moreover, they enhanced the growth parameters compared with the control. Our results proved that application of different biocontrol agents (P. fluorescens, P. lilacinus and P. guilliermondii) not only has a lethal effect on nematode, but also enhances the plant growth, supplying many nutritional elements and induction the systemic resistance in plants. Presence of C. parietina as a soil inhabitant cyanobacterium could antagonize biocontrol agents leading to the reduction of their practical efficiency in soil.     

Boiling water treatment: A simple,rapid and effective technique for nematode and banana weevil management in banana and plantain (Musa spp.) planting material

We studied the effect of treating banana and plantain (Musa spp.) suckers using boiling water for the generation of healthy planting material. Small, medium and large-sized suckers of two East African highland bananas (cvs. Mbwazirume, Kibuzi, AAA-EA) and one of plantain (cv. Agbagba, AAB) were dipped in boiling water for 10, 20 or 30 s after root removal and corm (sucker) paring. Treatments were compared with a conventional farmer method (control), which was not pared or treated with boiling water, and also with pared only. At three months after treatment, germination, plant height and girth of plants were similar within sucker size and between treatments for each cultivar. Root health was improved by boiling water treatment for all cultivars compared to untreated controls. Boiling water treatment of suckers reduced combined nematode densities of Helicotylenchus multicinctus, Radopholus similis and Meloidogyne spp. to 0.7% of farmer controls. Boiling water-treated suckers had less banana weevil damage than controls. Treatment of pared suckers for at least 20 s and up to 30 s was highly efficient for disinfesting banana and plantain planting material. This modification of the hot water treatment is a practical adaptation, suitable for smallholder growers to improve the quality of banana and plantain planting material in smallholder farms.     

基于环介导等温扩增技术检测江苏省中部地区小麦赤霉病病菌的种群组成

为明确江苏省中部地区小麦赤霉病病菌的种群组成,于2017年在江苏省镇江市、扬州市和南京市采集小麦赤霉病样本151份,采用可特异性识别亚细亚镰孢、禾谷镰孢、黄色镰孢、藤仓镰孢、燕麦镰孢、木贼镰孢和拟轮枝镰孢的环介导等温扩增（loop-mediated isothermal amplification,LAMP）检测技术检测了样本中该7种镰孢菌的分布情况。结果表明,从151份样本中共检测出7种镰孢菌中的6种。其中,亚细亚镰孢为优势种,检出率达100%;藤仓镰孢和禾谷镰孢为次优势种,检出率分别为10%和6%;黄色镰孢、燕麦镰孢和拟轮枝镰孢检出率依次为2.6%、2.0%和0.6%。小麦赤霉病存在复合侵染现象,从单份发病样本中最多检测到3种致病菌。LAMP法可用于小麦赤霉病病菌与种群组成的快速检测。     

High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea

Sclerotinia rot caused by the fungus Sclerotinia sclerotiorum is one of the most serious and damaging diseases of oilseed rape and there is keen worldwide interest to identify Brassica genotypes with resistance to this pathogen. Complete resistance against this pathogen has not been reported in the field, with only partial resistance being observed in some Brassica genotypes. Introgression lines were developed following hybridization of three wild crucifers (viz. Erucastrum cardaminoides, Diplotaxis tenuisiliqua and E. abyssinicum) with B. napus or B. juncea. Their resistance responses were characterized by using a stem inoculation test. Seed of 54 lines of B. napus and B. juncea obtained from Australia, India and China through an Australian Centre for International Agricultural Research (ACIAR) collaboration programme were used as susceptible check comparisons. Introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum had much higher levels (P < 0.001) of resistance compared with the ACIAR germplasm. Median values of stem lesion length of introgression lines derived from the wild species were 1.2, 1.7 and 2.0 cm, respectively, as compared with the ACIAR germplasm where the median value for stem lesion length was 8.7 cm. This is the first report of high levels of resistance against S. sclerotiorum in introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum. The novel sources of resistance identified in this study are a highly valuable resource that can be used in oilseed Brassica breeding programmes to enhance resistance in future B. napus and B. juncea cultivars against Sclerotinia stem rot.     

Phytotoxicity of Salvia spp. exudates

A study on thirteen Salvia species about the phytotoxicity of their aerial part exudates is reported. The selected species were: Salvia namaensis Schinz (1), Salvia fallax Fernald (2), Salvia disermas L. (3), Salvia chamaedryoides Cav. (4), Salvia confertiflora Pohl. (5), S. x jamensis J.Compton (6), Salvia buchananii Hedge (7), S. wagneriana Polak (8), Salvia scabra Linn.fil. (9), Salvia miniata Fernald (10), Salvia cacaliaefolia Benth.(11), Salvia adenophora Fernald (12), Salvia rutilans Carrière (13). For each species the inhibitory potential against Papaver rhoeas L. and Avena sativa L. was evidenced by dose-response studies and growth experiments performed both in Petri dish and in pot and also evaluated by means of germination indices and growth parameters. New indices useful for describing the phytotoxic response by a reduced number of testing solutions are proposed. Principal Component Analysis (PCA) of the germination data revealed that the exudate concentrations most significant in explaining the data variability were 1 and 2 μg/mL in Petri dish and 10 and 20 μg/mL in pot experiments. Root length and seedling weight were more important with respect to the other growth parameters.     

Mycoparasitic Trichoderma viride as a biocontrol agent against Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes and as a growth promoter of soybean

Trichoderma viride was proved as an effective biocontrol agent against two fungal pathogens, Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes, infecting soybean. During an in vitro biocontrol test, Trichoderma showed mycoparasitism and destructive control against the tested fungal pathogens. Both the pathogens significantly influence the germination and P. arrhenomanes had a severe effect (only 5% germination). The root system of the soybean plant was poorly developed due to the infection and it exerted a negative influence on the nodulation and further growth phases of the plant. During pot assay along with biocontrol activity, Trichoderma showed growth promoting action on the soybean plant. Trichoderma enhanced growth of shoot and root systems and fruit yield after 12 weeks of growth. Pythium and Fusarium infected plants treated with Trichoderma had ∼194% and 141% more height than pathogens alone. The fruit yield treated with Trichoderma was ∼66 per plant whereas the yield was only 41 for a control plant. The plants infected with Pythium and Fusarium and treated with Trichoderma had fruit yields of 43 and 53 respectively and those were 5 and 1.6 times higher than plants infected with pathogens.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Effects of crude seed extracts of Annona atemoya and Annona squamosa L. against the cabbage looper, Trichoplusia ni in the laboratory and greenhouse

Antifeedant, growth inhibitory and toxic effects of crude seed extracts of Annona squamosa and Annona atemoya from Fazenda Viveiro Bona, Parasisópolis – Minas Gerais, Brazil, were evaluated against the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) using different bioassays. Crude methanolic seed extracts deterred feeding of third instar T. ni larvae in a leaf disc choice bioassay. A. squamosa was ∼10 times more active as a feeding deterrent than A. atemoya (DC₅₀ = 2.3 mg/ml vs. 20.1 mg/ml). A. squamosa was ∼three times more active as a growth inhibitor than A. atemoya (EC₅₀ = 38.0 ppm vs. 117.0 ppm). Methanolic seed extracts of A. squamosa and A. atemoya were toxic to third instar T. ni larvae both through topical and oral application. A. squamosa was more toxic through feeding (LC₅₀ = 167.5 ppm vs. 382.4 ppm) whereas, A. atemoya exerted greater toxicity via topical application (LC₅₀ = 301.3 μg/larva vs. 197.7 μg/larva). Both A. squamosa and A. atemoya extracts reduced leaf area consumption and larval growth in a greenhouse experiment. Our results indicate that both A. squamosa and A. atemoya have potential for development as botanical insecticides, especially for local use in Brazil.     

Genetic diversity, population structure and relationships of Tunisian Thymus algeriensis Boiss. et Reut. and Thymus capitatus Hoffm. et Link. assessed by isozymes

The genetic diversity and population structure of two Tunisian Thymus species (Thymus algeriensis and Thymus capitatus), from 46 natural populations growing in six bioclimates, were analysed by starch gel electrophoresis using eight isozymes. The genetic diversity within populations varied at species level. Variation in T. algeriensis was higher than that observed for T. capitatus, and exclusive alleles were detected for the two studied species. A high differentiation among populations, for each species, estimated by Wright's F-statistics was revealed. In each species, a high level of inbreeding within populations induced by Wahlund effect was observed. A relatively high level of differentiation associated with a restricted gene flow among species was revealed. The PCA and UPGMA analysis, performed on all populations, showed two distinct groups with respect of specific differentiation level. The high genetic divergence between the two species corroborates their taxonomic status, as previously reported using morphological traits. The strategy for the management and conservation of populations should be made for each taxa according to its level of diversity and bioclimate.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.     

Nematicidal efficacy of isothiocyanates against root-knot nematode Meloidogyne javanica in cucumber

Isothiocyanates (ITCs), a series of new nematicides of the -NCS group, were evaluated for their efficacy against root-knot nematode Meloidogyne javanica. Of the compounds tested, AllylITC, AcITC, EtITC, BzTC, BzITC, 1-PEITC and 2-PEITC showed in vitro irreversible nematicidal activity against second-stage juveniles of M. javanica, following exposure for 72 h at concentrations as low as 5 μg mL⁻¹. When exposed to AllylITC, AcITC and EtITC at lower concentrations, motile juveniles also became irreversibly immobile in 3 days, with a LC₅₀ value at 2.76, 2.53 and 3.05 μg mL⁻¹, respectively. In the pot experiments, 1.0 ml AllylITC and 1.1 ml AcITC per kg of soil controlled M. javanica, similarly to or better than metam sodium at its recommended dose. Similar results were obtained in the field experiments using 1.0 kg AllylITC or 1.0 kg AcITC ha⁻¹. Based on the results of this study, AllylITC and AcITC have potential to be used as new bio-fumigant nematicides.     

Field evaluation of yield effects on the U.S.A. heirloom sweetpotato cultivars infected by Sweet potato leaf curl virus

The incidence of Sweet potato leaf curl virus (SPLCV), a Begomovirus, on sweetpotato, Ipomoea batatas (L.) Lam. (Convolvulaceae), in South Carolina, U.S.A. has increased rapidly in recent years. This is likely due to the use of infected propagating materials and the increasing population of its vector, the sweetpotato whitefly, Bemisia tabaci (Gennadius). In this study, we demonstrated in field experiments that SPLCV infection reduced the yields of most heirloom sweetpotato lines relative to the yields of non-infected plants. Real-time polymerase chain reaction (PCR) technology specific to several common sweetpotato viruses was used to determine the virus infection status in 69 selected accessions of heirloom sweetpotato lines. Meristem tip culture technology was used to regenerate virus-free plants from these materials. To ensure the virus-free status, each mericlone was evaluated using real-time PCR and graft bio-indexing on the indicator species, Ipomoea setosa Ker Gawl. Mericlones of 27 cultivars were found to be free of the viruses. The 27 cultivars were included in a field test to determine the effect of SPLCV infection on yield. Yields of virus-free plants of the cultivars ranged from 10 to 80% greater than the yields of SPLCV-infected plants. However, the yield differences between virus-free and infected plants were diminished in the second year of the field experiment due to a rapid re-infection by SPLCV. These results demonstrate the importance of using certified, virus-tested seed roots or cuttings. The rapid re-infection of the virus-tested sweetpotato plants with SPLCV observed in these studies suggests that management of the whitefly population should be a critical element in control of this important virus.     

Integrated management of Striga hermonthica, stemborers,and declining soil fertility in western Kenya

Striga hermonthica (Delile) Benth., stemborers, and declining soil fertility are serious threats to sustainable food production in the Lake Victoria zone of Kenya. To address these constraints, promising integrated crop management technologies were evaluated, using a multi-locational design in four sub-locations in Siaya and Vihiga district (western Kenya) for six cropping seasons. Technologies evaluated consisted of the traditional maize (Zea mays L.) – bean (Phaseolus vulgaris L.) intercrop, maize – Desmodium (Desmodium uncinatum (Jacq.) DC.) push–pull intercrop, Crotalaria (Crotalaria ochroleuca G. Don) – maize rotation, and soybean (Glycine max (L.) Merr) – maize rotation. Within each of these systems, imazapyr-coated herbicide-resistant maize (IR-maize) and fertilizer were super-imposed as sub-plot factors. The push–pull system was observed to significantly reduce Striga emergence and stemborer damage from the second season onwards. IR-maize reduced and delayed Striga emergence from the first cropping season. Differences in Striga emergence and stemborer damage between the other systems were not significantly different. After five cropping seasons, the Striga seedbank was significantly higher in the maize-bean intercrop system than in the push–pull system under both maize varieties while the rotational systems had intermediate values not different from the day zero values. Under IR-maize, the Striga seedbank was significantly lower than under local maize for all cropping systems. Maize yields varied between seasons, districts, and cropping systems. Yields in the push–pull system were higher than in the maize-bean intercrop after two seasons and in the absence of mid-season drought stress. Both maize and soybean responded significantly to fertilizer application for both districts and for most seasons. The various interventions did not substantially affect various soil fertility-related parameters after five seasons. In the short term, IR-maize integrated in a push–pull system is the most promising option to reduce Striga while the rotational systems may need a longer timeframe to reduce the Striga seedbank. Finally, farmer-led evaluation of the various technologies will determine which of those is really most acceptable under the prevailing farming conditions.     

Mature watermelon vine decline: Suppression with fumigants of a soil-borne problem and association with Rhizopycnis vagum

Mature watermelon vine decline (MWVD) is a late-season disease of unknown etiology, characterized by vine collapse and discolored, much reduced root systems. While several new disorders in watermelon have been reported recently, this problem appears specific to the Midwest. To test for a biological cause and methods to suppress this disorder, soil was collected from two southern Indiana fields with histories of MWVD for microplot and greenhouse experiments. Watermelons grew best in microplots treated with methyl bromide or methyl iodide. Roots of plants growing in the untreated MWVD soil were severely necrotic; those from the fumigation treatments appeared healthy. In heating experiments, watermelon seedlings were grown in autoclaved soil mixed with 10% MWVD soil that was either untreated or heated at 40, 50, 60, 70 or 80 °C for 30 min. Watermelon plants in the untreated controls and the 40 and 50 °C treatments had reduced top dry weights with decreased fibrous roots and associated discoloration in contrast to treatments of equal to and greater than 60 °C. Representative cultures from necrotic roots were identified as Rhizopycnis vagum by morphological analysis and sequencing the ITS region of the ribosomal DNA. PCR analysis with specific primers confirmed that R. vagum was present in roots from untreated MWVD soil but not in symptomless roots. This is the first report of R. vagum associated with plants suffering from MWVD in Indiana and indicates that this fungus occurs much farther north in the U.S. than known previously.     

Combined use of Streptomyces sp. A6 and chemical fungicides against fusarium wilt of Cajanus cajan may reduce the dosage of fungicides required in the field

Biological control agents offer one of the best alternatives to reduce the use of pesticides. This investigation studied the tolerance to fungicides and integrated use of the potential biocontrol agent Streptomyces sp. A6 for control of Fusarium wilt of pigeon pea, Cajanus cajan. Streptomyces sp. A6 exhibited strong tolerance towards most of the fungicides used in the study at concentrations higher than those recommended for field applications. The isolate showed enhanced growth and mycolytic enzyme production in the presence of sulphur, mancozeb, carbendazim, fosetyl aluminium and triadimefon. The fungicides mancozeb, sulphur and carbendazim were selected for further studies. Effective concentrations (EC₅₀ values) of the test fungicides that reduced Fusarium spore germination and fungal biomass by 50% were determined. Similarly, the EC₅₀ for inhibiting fungal spore germination and reducing fungal biomass to 50% by Streptomyces sp. A6 and culture filtrate (CF) were also determined. Combining the EC₅₀ dose of the culture and CF with test fungicides was found to be more effective for controlling Fusarium infection in C. cajan compared to the sum of the effects of the individual treatments. Such combined use of biocontrol agent with fungicides can reduce the dosage of toxic fungicides in agricultural fields, thereby reducing environmental risks. Tolerance and synergistic interaction of Streptomyces sp. A6 with frequently used fungicides suggested its potential in integrated pest management. To the best our knowledge, this is the first extensive study on integrated use of Streptomyces species with fungicides.     

Population dynamics of Xanthomonas campestris pv. vitians on different plant species and management of bacterial leaf spot of lettuce under greenhouse conditions

The population dynamics of Xanthomonas campestris pv. vitians (Xcv) was studied both externally and internally in lettuce, tomato and pepper plants. In addition, the application of bactericides during transplant production period was carried out for the management of bacterial leaf spot of lettuce under greenhouse conditions. Epiphytic populations of Xcv were recovered on leaves of lettuce plants (10⁵ CFU/g) 5 weeks after sprayed than the other plant species when inoculated with 10⁸ CFU/ml of Xcv. When plants of each crop species infiltrated with the bacterium at 10⁵ CFU/ml, the highest populations were developed in lettuce (10⁸ CFU/cm²) followed by pepper with 10⁶ CFU/cm² and tomato with 10⁵ CFU/cm² 10-days after infiltration. Application of a mixture of Maneb and Kocide or Kocide alone as a foliar spray on lettuce significantly reduced the incidence and disease severity of bacterial leaf spot by 29 and 27% respectively. Spread of the bacterium and development of the disease may partly be managed by avoiding intercropping of these plants commonly grown in close proximity to lettuce. For the management of leaf-associated populations of Xcv in lettuce, use of a mixture of Maneb and Kocide is advocated to minimize the effect of attacks.